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DNA methylation in demyelinated multiple sclerosis hippocampus Anthony M. Chomyk1, Christina Volsko1, Ajai Tripathi1, Sadie A. Deckard1, Bruce D. Trapp1, Robert J. Fox2 & Ranjan Dutta1
Received: 3 February 2017 Accepted: 10 July 2017 Published: xx xx xxxx
Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the human central nervous system (CNS). Memory impairments and hippocampal demyelination are common features in MS patients. Our previous data have shown that demyelination alters neuronal gene expression in the hippocampus. DNA methylation is a common epigenetic modifier of gene expression. In this study, we investigated whether DNA methylation is altered in MS hippocampus following demyelination. Our results show that mRNA levels of DNA methyltransferase were increased in demyelinated MS hippocampus, while de-methylation enzymes were decreased. Comparative methylation profiling identify hypo-methylation within upstream sequences of 6 genes and hyper-methylation of 10 genes in demyelinated MS hippocampus. Genes identified in the current study were also validated in an independent microarray dataset generated from MS hippocampus. Independent validation using RTPCR revealed that DNA methylation inversely correlated with mRNA levels of the candidate genes. Queries across cell-specific databases revealed that a majority of the candidate genes are expressed by astrocytes and neurons in mouse and human CNS. Taken together, our results expands the list of genes previously identified in MS hippocampus and establish DNA methylation as a mechanism of altered gene expression in MS hippocampus. Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) that affects more than two million people worldwide1, 2. Among the spectrum of cognitive impairments, memory dysfunction is most common among MS patients3, 4. Hippocampal demyelination is extensive in individuals with MS and modulates expression of neuronal genes involved in synaptic plasticity and memory function5, 6. Large-scale genome-wide association studies (GWAS) have identified MS susceptibility loci, including human leukocyte antigen loci and other immune-function related genes7–10; however, their functional significance related to MS pathogenesis is still unknown. The relatively low concordance rate of single-nucleotide polymorphisms in monozygotic twins11, the presence of a strong gender bias, and the influence of migration on disease onset collectively suggest that the pathogenesis of MS likely results from a combination of both genetic and epigenetic factors12, 13. Epigenetic modifications, including DNA methylation, histone modification, chromatin remodeling, and noncoding RNA regulation have been reported to regulate gene expression and to participate in the etiology of MS14, 15. DNA methylation occurs in special genomic regions called CpG islands, which contain greater than 50% cytosine and guanine nucleotides. It plays a major role in aberrant expression of genes that are important in several neurological diseases16, 17 as well as in memory formation and maintenance18, 19. We previously compared and identified several genes and microRNAs in MS hippocampus that correlate with synaptic changes, memory dysfunction, and hippocampal demyelination5, 20. In this study, we investigated additional epigenetic mechanisms that alter gene expression in MS hippocampus. We found significant increases in mRNA levels of key DNA methyltransferase enzymes (DNMTs). Interestingly, the mRNA levels of the three DNA de-methylation enzymes (ten-eleven translocation methylcytosine dioxygenase 1–3; TET1-TET3), which catalyze hydroxy-methylation as well as the total level of hydroxy-methylated residues, were significantly decreased in MS demyelinated hippocampus. Several differentially methylated positions (DMPs) were also identified by comparing MS myelinated to demyelinated hippocampus. The methylation status of DMPs inversely correlated with mRNA levels of target genes that have been associated with neuronal survival and memory function. As methylation patterns in different cell types may contribute to overall methylation 1 Department of Neurosciences, Cleveland Clinic, Cleveland, OH, 44195, USA. 2Mellen Center for MS Research, Cleveland Clinic, Cleveland, OH, 44195, USA. Correspondence and requests for materials should be addressed to R.D. (email: [email protected])
Scientific REPOrTS | 7: 8696 | DOI:10.1038/s41598-017-08623-5
1
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Figure 1. DNA methyltransferase (DNMT) expression in hippocampi from multiple sclerosis (MS) brains. RT-PCR analysis shows significant increases in mRNA levels of DNMT 1, DNMT3A, and DNMT3B in demyelinated hippocampus (n = 4) compared to myelinated hippocampus (n = 4) (A). Immunohistochemistry showing cellular expression of DNMT1, DNMT3A, and DNMT3B in myelinated (B,D,F) and demyelinated hippocampus (C,E,G), with predominant expression in hippocampal neurons. Scale Bars: B–G: 30 μm; Error bars indicate + S.E.M.; *p
OPEN
DNA methylation in demyelinated multiple sclerosis hippocampus Anthony M. Chomyk1, Christina Volsko1, Ajai Tripathi1, Sadie A. Deckard1, Bruce D. Trapp1, Robert J. Fox2 & Ranjan Dutta1
Received: 3 February 2017 Accepted: 10 July 2017 Published: xx xx xxxx
Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the human central nervous system (CNS). Memory impairments and hippocampal demyelination are common features in MS patients. Our previous data have shown that demyelination alters neuronal gene expression in the hippocampus. DNA methylation is a common epigenetic modifier of gene expression. In this study, we investigated whether DNA methylation is altered in MS hippocampus following demyelination. Our results show that mRNA levels of DNA methyltransferase were increased in demyelinated MS hippocampus, while de-methylation enzymes were decreased. Comparative methylation profiling identify hypo-methylation within upstream sequences of 6 genes and hyper-methylation of 10 genes in demyelinated MS hippocampus. Genes identified in the current study were also validated in an independent microarray dataset generated from MS hippocampus. Independent validation using RTPCR revealed that DNA methylation inversely correlated with mRNA levels of the candidate genes. Queries across cell-specific databases revealed that a majority of the candidate genes are expressed by astrocytes and neurons in mouse and human CNS. Taken together, our results expands the list of genes previously identified in MS hippocampus and establish DNA methylation as a mechanism of altered gene expression in MS hippocampus. Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) that affects more than two million people worldwide1, 2. Among the spectrum of cognitive impairments, memory dysfunction is most common among MS patients3, 4. Hippocampal demyelination is extensive in individuals with MS and modulates expression of neuronal genes involved in synaptic plasticity and memory function5, 6. Large-scale genome-wide association studies (GWAS) have identified MS susceptibility loci, including human leukocyte antigen loci and other immune-function related genes7–10; however, their functional significance related to MS pathogenesis is still unknown. The relatively low concordance rate of single-nucleotide polymorphisms in monozygotic twins11, the presence of a strong gender bias, and the influence of migration on disease onset collectively suggest that the pathogenesis of MS likely results from a combination of both genetic and epigenetic factors12, 13. Epigenetic modifications, including DNA methylation, histone modification, chromatin remodeling, and noncoding RNA regulation have been reported to regulate gene expression and to participate in the etiology of MS14, 15. DNA methylation occurs in special genomic regions called CpG islands, which contain greater than 50% cytosine and guanine nucleotides. It plays a major role in aberrant expression of genes that are important in several neurological diseases16, 17 as well as in memory formation and maintenance18, 19. We previously compared and identified several genes and microRNAs in MS hippocampus that correlate with synaptic changes, memory dysfunction, and hippocampal demyelination5, 20. In this study, we investigated additional epigenetic mechanisms that alter gene expression in MS hippocampus. We found significant increases in mRNA levels of key DNA methyltransferase enzymes (DNMTs). Interestingly, the mRNA levels of the three DNA de-methylation enzymes (ten-eleven translocation methylcytosine dioxygenase 1–3; TET1-TET3), which catalyze hydroxy-methylation as well as the total level of hydroxy-methylated residues, were significantly decreased in MS demyelinated hippocampus. Several differentially methylated positions (DMPs) were also identified by comparing MS myelinated to demyelinated hippocampus. The methylation status of DMPs inversely correlated with mRNA levels of target genes that have been associated with neuronal survival and memory function. As methylation patterns in different cell types may contribute to overall methylation 1 Department of Neurosciences, Cleveland Clinic, Cleveland, OH, 44195, USA. 2Mellen Center for MS Research, Cleveland Clinic, Cleveland, OH, 44195, USA. Correspondence and requests for materials should be addressed to R.D. (email: [email protected])
Scientific REPOrTS | 7: 8696 | DOI:10.1038/s41598-017-08623-5
1
www.nature.com/scientificreports/
Figure 1. DNA methyltransferase (DNMT) expression in hippocampi from multiple sclerosis (MS) brains. RT-PCR analysis shows significant increases in mRNA levels of DNMT 1, DNMT3A, and DNMT3B in demyelinated hippocampus (n = 4) compared to myelinated hippocampus (n = 4) (A). Immunohistochemistry showing cellular expression of DNMT1, DNMT3A, and DNMT3B in myelinated (B,D,F) and demyelinated hippocampus (C,E,G), with predominant expression in hippocampal neurons. Scale Bars: B–G: 30 μm; Error bars indicate + S.E.M.; *p
